The principal function of the urea cycle is the conversion of toxic ammonia into the relatively non-toxic metabolite, urea. Many interrelationships exist between the urea cycle and other metabolic pathways. Thus, multiple regulatory points are probably required for the efficient functioning of the pathway. Carbamylphosphate synthetase I (CPS I) is ideally located in the mitochondrial matrix to regulate the flux of precursors into the urea cycle. In rat liver, CPS I activity is modulated by N-acetyl-L-glutamate (AG), which serves as an allosteric activator. N-acetyl-L-glutamate synthetase (AGS) can potentially regulate CPS I by controlling the accumulation of AG. AGS activity is modulated by the presence of L-arginine. Virtually no comprehensive study of CPS I from human liver has been conducted. AGS has not been thoroughly characterized from any source; the enzyme level has not been measured in human liver. We intend to evaluate the regulatory role of both CPS I and AGS in urea biosynthesis in human liver. The elucidation of their regulatory function(s) can best be achieved by a thorough biochemical and regulatory study. CPS I and AGS will be purified from healthy human liver by standard biochemical techniques. This will allow for the determination of molecular weight, sedimentation properties, subunit analysis, amino acid composition, and kinetic parameters of both enzymes. An intensive effort will be exerted to define the regulatory properties of both CPS I and AGS. This will include investigations into the possible protein-protein and protein-ligand interactions of these enzymes.